Heterocyclic Compounds

ABSTRACT

Certain thienopyrrolyl and furanopyrrolyl compounds are disclosed as useful to treat or prevent disorders and conditions mediated by the histamine H 4  receptor, including allergic rhinitis.

This application is a division of U.S. patent application Ser. No.10/656,059, filed Sep. 5, 2003, which in turn claims the benefit of U.S.provisional patent application Ser. No. 60/408,723, filed Sep. 6, 2002.

FIELD OF THE INVENTION

The invention relates to novel, pharmaceutically active, fusedheterocyclic compounds and methods of using them to treat or preventdisorders and conditions mediated by the histamine H₄ receptor.

BACKGROUND OF THE INVENTION

Histamine was first identified as a hormone (G. Barger and H. H. Dale,J. Physiol. (London) 1910, 41:19-59) and has since been demonstrated toplay a major role in a variety of physiological processes, including theinflammatory “triple response” via H₁ receptors (A. S. F. Ash and H. O.Schild, Br. J. Pharmac. Chemother. 1966, 27:427-439), gastric acidsecretion via H₂ receptors (J. W. Black et al., Nature 1972,236:385-390), and neurotransmitter release in the central nervous systemvia H₃ receptors (J.-M. Arrang et al., Nature 1983, 302:832-837) (forreview see S. J. Hill et al., Pharmacol. Rev. 1997, 49(3):253-278). Allthree histamine receptor subtypes have been demonstrated to be membersof the superfamily of G protein-coupled receptors (I. Gantz et al.,Proc. Natl. Acad. Sci. U.S.A. 1991, 88:429-433; T. W. Lovenberg et al.,Mol. Pharmacol. 1999, 55(6):1101-1107; M. Yamashita et al., Proc. Natl.Acad. Sci. U.S.A. 1991, 88:11515-11519). There are, however, additionalfunctions of histamine that have been reported, for which no receptorhas been identified. For example, in 1994, Raible et al. demonstratedthat histamine and R-α-methylhistamine could activate calciummobilization in human eosinophils (D. G. Raible et al., Am. J. Respir.Crit. Care Med. 1994,149:1506-1511). These responses were blocked by theH₃-receptor antagonist thioperamide. However, R-α-methylhistamine wassignificantly less potent than histamine, which was not consistent withthe involvement of known H₃ receptor subtypes. Therefore, Raible et al.hypothesized the existence of a novel histamine receptor on eosinophilsthat was non-H₁, non-H₂, and non-H₃. Most recently several groups (T.Oda et al., J. Biol. Chem. 2000, 275(47):36781-36786; C. Liu et al.,Mol. Pharmacol. 2001, 59(3):420-426; T. Nguyen et al., Mol. Pharmacol.2001, 59(3):427-433; Y. Zhu et al., Mol. Pharmacol. 2001, 59(3):434-441;K. L. Morse et al., J. Pharmacol. Exp. Ther. 2001, 296(3):1058-1066)have identified and characterized a fourth histamine receptor subtype,the H₄ receptor. This receptor is a 390 amino acid, seven-transmembrane,G protein-coupled receptor with approximately 40% homology to thehistamine H₃ receptor. In contrast to the H₃ receptor, which isprimarily located in the brain, the H₄ receptor is expressed at greaterlevels in neutrophils and mast cells, among other cells, as reported byMorse et al. (see above).

Events that elicit the inflammatory response include physicalstimulation (including trauma), chemical stimulation, infection, andinvasion by a foreign body. The inflammatory response is characterizedby pain, increased temperature, redness, swelling, reduced function, ora combination of these. Many conditions, such as allergies, asthma,chronic obstructed pulmonary disease (COPD), atherosclerosis, andautoimmune diseases, including rheumatoid arthritis and lupus, arecharacterized by excessive or prolonged inflammation. Inhibition ofleukocyte recruitment can provide significant therapeutic value.Inflammatory diseases or inflammation-mediated diseases or conditionsinclude, but are not limited to, acute inflammation, allergicinflammation, and chronic inflammation.

Mast cell de-granulation (exocytosis) leads to an inflammatory responsethat may be initially characterized by a histamine-modulated wheal andflare reaction. A wide variety of immunological (e.g., allergens orantibodies) and non-immunological (e.g., chemical) stimuli may cause theactivation, recruitment, and de-granulation of mast cells. Mast cellactivation initiates allergic (H₁) inflammatory responses, which in turncause the recruitment of other effector cells that further contribute tothe inflammatory response. The histamine H2 receptors modulate gastricacid secretion, and the histamine H3 receptors affect neurotransmitterrelease in the central nervous system.

Examples of textbooks on the subject of inflammation include J. I.Gallin and R. Snyderman, Inflammation: Basic Principles and ClinicalCorrelates, 3^(rd) Edition, (Lippincott Williams & Wilkins,Philadelphia, 1999); V. Stvrtinova, J. Jakubovsky and 1. Hulin,“Inflammation and Fever”, Pathophysiology Principles of Diseases(Textbook for Medical Students, Academic Press, 1995); Cecil et al.,Textbook Of Medicine, 18^(th) Edition (W.B. Saunders Company, 1988); andSteadmans Medical Dictionary.

SUMMARY OF THE INVENTION

The invention features a compound of formula (I):

-   Y is O or S;-   Z is O or S;-   n is 1 or 2;-   m is 1 or 2;-   n+m is 2 or 3;-   R¹ is H or C₁₋₆alkyl;-   R² is H, F, Cl, Br or C₁₋₆alkyl;-   R³ and R⁴ are, independently, H, C₁₋₄alkyl, C₃₋₆cycloalkyl,    C₁₋₄alkyl(C₃₋₆cycloalkyl), cyano, —CF₃, —(CO)NR^(p)R^(q),    —(CO)OR^(r), —CH₂NR^(p)R^(q) Or —CH₂OR^(r); where R^(p), R^(q) and    R^(r) are independently selected from H, C₁₋₄alkyl, C₃₋₆cycloalkyl,    phenyl, —C₁₋₂alkyl(C₃₋₆cycloalkyl), benzyl or phenethyl, or R^(p)    and R^(q) taken together with the nitrogen to which they are    attached, form a 4-7 membered heterocyclic ring with 0 or 1    additional heteroatoms selected from O, S, NH or NC₁₋₆alkyl, and    where any phenyl or alkyl or cycloalkyl moiety of the foregoing is    optionally and independently substituted with between 1 and 3    substituents selected from C₁₋₃alkyl, halo, hydroxy, amino, and    C₁₋₃alkoxy;-   R⁵ and R⁶ are, independently, H or C₁₋₆alkyl;-   R⁷ is —R^(a), —R^(b)R^(a), —R^(e)—R^(a) or —R^(e)—N(R^(c))(R^(d)),    where R^(a) is H, cyano, —(C═O)N(R^(c))(R^(d)), —C(═NH)(NH₂),    C₁₋₁₀alkyl, C₂₋₈alkenyl, C₃₋₈cycloalkyl, C₄₋₇heterocyclic radical or    phenyl, where the C₄₋₇heterocyclic radical is attached at a carbon    atom and contains one of O, S, NH or NC₁₋₄alkyl, and optionally an    additional NH or NC₁₋₆alkyl in rings of 5 or 6 or 7 members, where    R^(b) is C₁₋₈alkylene or C₂₋₈alkenylene, where R^(e) is C₂₋₈alkylene    or C₂₋₈alkenylene, where R^(c) and R^(d) are each independently H,    C₁₋₄alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkyl or phenyl, or R^(c) and R^(d)    taken together with the nitrogen to which they are attached, form a    4-7 membered heterocyclic ring with 0 or 1 additional heteroatoms    selected from O, S, NH or NC₁₋₆alkyl, and where any phenyl or alkyl    or cycloalkyl moiety of the foregoing is optionally and    independently substituted with between 1 and 3 substituents selected    from C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy;    -   alternatively, R⁷ may be taken together with an adjacent R⁴ as        well as their carbon and nitrogen of attachment to form a 5, 6        or 7 membered heterocyclic ring, with 0 or 1 additional        heteroatoms selected from O, S, NH or NC₁₋₆alkyl, and optionally        and independently substituted with between 1 and 3 substituents        selected from C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy;-   R⁸ and R⁹ are, independently, H, F, Cl, Br, I, C₁₋₄alkyl,    C₁₋₄alkoxy, —C₃₋₆cycloalkyl, —OC₃₋₆cycloalkyl, —OCH₂Ph, —CF₃, —OCF₃,    —SCF₃, —(C═O)R^(k) (wherein R^(k) is H, C₁₋₄alkyl, —OH, phenyl,    benzyl, phenethyl or C₁₋₆alkoxy), —(N—R^(t))(C═O)R^(k) (where R^(t)    is H or C₁₋₄alkyl), —(N—R^(t))SO₂C₁₋₄alkyl, —(S═(O)_(p))—C₁₋₄alkyl    (wherein p is 0, 1 or 2), nitro, —SO₂NR^(l)R^(m) (wherein R^(l) and    R^(m) are independently selected from H, C₁₋₄alkyl, phenyl, benzyl    or phenethyl, or R^(l) and R^(m) taken together with the nitrogen to    which they are attached, form a 4-7 membered heterocyclic ring with    0 or 1 additional heteroatoms selected from O, S, NH or NC₁₋₄alkyl),    —(C═O)NR^(l)R^(m), cyano or phenyl, where any phenyl or alkyl or    cycloalkyl moiety of the foregoing is optionally and independently    substituted with between 1 and 3 substituents selected from    C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy;    and enantiomers, diastereomers and pharmaceutically acceptable salts    and esters thereof,    with the following provisos,    that R⁶ adjacent to N must be H where R⁴ adjacent to N is other than    H,    that R⁷ is not —CH₂CH₂OH; and    that where the core molecule is a 4H-furo, then one of R⁴ and R⁶    adjacent to N must not be methyl when the other is hydrogen unless    R⁶ and R⁴ are taken together to form a bridging moiety.

The invention also features pharmaceutical compositions containing suchcompounds and methods of using such compositions in the treatment orprevention of H₄-mediated diseases and conditions, particularly thosewherein it is desirable to antagonize the H₄ receptor.

DETAILED DESCRIPTION

Preferably, Y is S.

Preferably, Z is O.

Preferably, n is 1 and m is 1.

Preferrably, R¹ is selected from the group consisting of H or methyl.

Preferrably, R² is H.

Preferrably, R³ and R⁴ are, independently, selected from the groupconsisting of

a) H,

b) —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, n-butyl, i-butyl, t-butyl,

c) cyclopropyl, cyclopentyl, cyclohexyl, —CH₂cyclopropyl,—CH₂cyclopentyl, —CH₂cyclohexyl, —CH₂Ocyclopropyl, —CH₂Ocyclopentyl,—CH₂Ocyclohexyl,

d) cyano,

e) trifluoromethyl,

f) —(C═O)NH₂, —(C═O)NHC₁₋₄alkyl, —(C═O)N(C₁₋₄alkyl)₂, —(C═O)NHphenyl,—(C═O)pyrrolidin-1-yl, —(C═O)imidazolidin-1-yl, —(C═O)pyrazolidin-1-yl,(C═O)piperidin-1-yl, —(C═O)piperazin-1-yl, —(C═O)morpholin-4-yl,—(C═O)thiomorpholin-4-yl,

g) —COOH, —COOCH₃, —COOCH₂CH₃, —COOphenyl, —COObenzyl,

h) —CH₂NH₂, —CH₂NHC₁₋₄alkyl, —CH₂N(C₁₋₄alkyl)₂, —CH₂NHphenyl,—CH₂NHbenzyl, —CH₂pyrrolidin-1-yl, —CH₂imidazolidin-1-yl,—CH₂pyrazolidin-1-yl, —CH₂piperidin-1-yl, —CH₂piperazin-1-yl,—CH₂morpholin-4-yl, —CH₂thiomorpholin-4-yl,

i) —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂OCH₃, —CH₂OCH₂CH₃,—CH₂OCH₂CH₂CH₃, —CH₂OCH(CH₃)₂, —CH₂O-n-butyl, —CH₂O-1-butyl,—CH₂O-t-butyl, —CH₂O phenyl, —CH₂Obenzyl and —CH₂OCH₂cyclopropyl.

Most preferrably, R³ and R⁴ are, independently, H or —CH₃.

Preferrably, R⁵ and R⁶ are, independently, selected from the groupconsisting of H and methyl.

Most preferrably, R⁵ and R⁶ are H.

Preferrably, R⁷ is selected from the group consisting of

a) H, —CH₂CH₂CH₂OH,

b) cyano,

c) —(C═O)NH₂, —(C═O)NHC₁₋₄alkyl, —(C═O)N(C₁₋₄alkyl)₂, —(C═O)NHphenyl,—(C═O)pyrrolidin-1-yl, —(C═O)imidazolidin-1-yl, —(C═O)pyrazolidin-1-yl,—(C═O)piperidin-1-yl, —(C═O)piperazin-1-yl, —(C═O)morpholin-4-yl,—(C═O)thiomorpholin-4-yl, —CH₂(C═O)NH₂, —CH₂(C═O)NHC₁₋₄alkyl,—CH₂(C═O)N(C₁₋₄alkyl)₂, —CH₂(C═O)NHphenyl, —CH₂(C═O)pyrrolidin-1-yl,—CH₂(C═O)imidazolidin-1-yl, —CH₂(C═O)pyrazolidin-1-yl,—CH₂(C═O)piperidin-1-yl, —CH₂(C═O)piperazin-1-yl,—CH₂(C═O)morpholin-4-yl, —CH₂(C═O)thiomorpholin-4-yl, —CH₂CH₂O(C═O)NH₂,—CH₂CH₂O(C═O)NHC₁₋₄alkyl, —CH₂CH₂O(C═O)N(C₁₋₄alkyl)₂,—CH₂CH₂O(C═O)NHphenyl, —CH₂CH₂O(C═O)pyrrolidin-1-yl,—CH₂CH₂O(C═O)imidazolidin-1-yl, —CH₂CH₂O(C═O)pyrazolidin-1-yl,—CH₂CH₂O(C═O)piperidin-1-yl, —CH₂CH₂O(C═O)piperazin-1-yl,—CH₂CH₂O(C═O)morpholin-4-yl, —CH₂CH₂O(C═O)thiomorpholin-4-yl,

d) —C(═NH)(NH₂), —CH₂C(═NH)(NH₂),

e) —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, n-butyl, i-butyl, t-butyl,—CH₂CH₂OCH₃, —CH₂CH₂OCH₂CH₃, —CH₂CH₂OCH₂CH₂CH₃, —CH₂CH₂OCH(CH₃)₂,—CH₂CH₂O-n-butyl, —CH₂CH₂O-1-butyl, —CH₂CH₂O-t-butyl,

f), —CH₂CH═CH₂,

g) cyclopropyl, cyclopentyl, cyclohexyl, —CH₂cyclopropyl,—CH₂cyclopentyl, —CH₂cyclohexyl, —CH₂CH₂Ocyclopropyl,—CH₂CH₂Ocyclopentyl, —CH₂CH₂Ocyclohexyl,

h) pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl,piperazinyl, morpholinyl, thiomorpholinyl, —CH₂pyrrolidinyl,—CH₂imidazolidinyl, —CH₂pyrazolidinyl, —CH₂piperidinyl, —CH₂piperazinyl,—CH₂morpholinyl, —CH₂thiomorpholinyl,

i) —CH₂CH₂NH₂, —CH₂CH₂NHC₁₋₄alkyl, —CH₂CH₂N(C₁₋₄alkyl)₂,—CH₂CH₂NHphenyl, —CH₂CH₂pyrrolidin-1-yl, —CH₂CH₂imidazolidin-1-yl,—CH₂CH₂pyrazolidin-1-yl, —CH₂CH₂piperidin-1-yl, —CH₂CH₂piperazin-1-yl,—CH₂CH₂morpholin-4-yl, —CH₂CH₂thiomorpholin-4-yl,

j) phenyl, benzyl, phenethyl and benzyloxymethyl.

Most preferrably, R⁷ is selected from the group consisting of H and—CH₃.

Preferred R⁷ taken together with an adjacent R⁴ as well as their carbonand nitrogen of attachment are pyrrolidin-1,2-yl, imidazolidin-1,2-yl,imidazolidin-1,5-yl, pyrazolidin-1,5-yl, piperidin-1,2-yl,piperazin-1,2-yl, morpholin-4,5-yl and thiomorpholin-4,5-yl.

Most preferred R⁷ taken together with an adjacent R⁴ as well as theircarbon and nitrogen of attachment are pyrrolidin-1,2-yl andpiperidin-1,2-yl.

Preferrably, R⁸ and R⁹ are, independently, selected from the groupconsisting of H, —F, —Cl, —Br, —I, —CH₃, —CH₂CH₃, —OCH₃, —OCH₂CH₃,—OCH(CH₃)₂, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,—Ocyclopentyl, —Ocyclohexyl, —CF₃, —OCF₃, —SCF₃, —COOH, —COOCH₃,—COOCH₂CH₃, —C(O)CH₃, —NHCOCH₃, —NCH₃COCH₃, —NHSO₂CH₃, —NCH₃SO₂CH₃,—SOCH₃, —SO₂CH₃, —NO₂, —SO₂NH₂, —SO₂NHCH₃, —SO₂N(CH₃)₂, —C(O)NH₂,—C(O)N(CH₃)₂, —C(O)NH(CH₃), —CN and phenyl.

Most preferrably, R⁸ and R⁹ are, independently, selected from the groupconsisting of hydrogen, methyl, chloro and bromo. Further, it is mostpreferred that one or both of R⁸ and R⁹ are not hydrogen.

The “pharmaceutically acceptable salts and esters thereof” refer tothose salt and ester forms of the compounds of the present inventionthat would be apparent to the pharmaceutical chemist, i.e., those thatare non-toxic and that would favorably affect the pharmacokineticproperties of said compounds of the present invention. Those compoundshaving favorable pharmacokinetic properties would be apparent to thepharmaceutical chemist, i.e., those that are non-toxic and that possesssuch pharmacokinetic properties to provide sufficient palatability,absorption, distribution, metabolism and excretion. Other factors, morepractical in nature, that are also important in the selection are costof raw materials, ease of crystallization, yield, stability,hygroscopicity, and flowability of the resulting bulk drug. In addition,acceptable salts of carboxylates include sodium, potassium, calcium andmagnesium. Examples of suitable cationic salts include hydrobromic,hydroiodic, hydrochloric, perchloric, sulfuric, maleic, fumaric, malic,tartatic, citric, benzoic, mandelic, methanesulfonic,hydroethanesulfonic, benzenesulfonic, oxalic, pamoic,2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic andsaccharic. Examples of suitable esters include such esters where one ormore carboxyl substituents is replaced with p-methoxybenzyloxycarbonyl,2,4,6-trimethylbenzyloxycarbonyl, 9-anthryloxycarbonyl, CH₃SCH₂COO—,tetrahydrofur-2-yloxycarbonyl, tetrahydropyran-2-yloxycarbonyl,fur-2-uloxycarbonyl, benzoyl methoxycarbonyl, p-nitrobenzyloxycarbonyl,4-pyridyl methoxycarbonyl, 2,2,2-trichloroethoxycarbonyl,2,2,2-tribromoethoxycarbonyl, t-butyloxycarbonyl, t-amyloxycarbonyl,diphenylmethoxycarbonyl, triphenylmethoxycarbonyl, adamantyloxycarbonyl,2-benzyloxyphenyloxycarbonyl, 4-methylthiophenyloxycarbonyl, ortetrahydropyran-2-yloxycarbonyl.

The provisos are based on a failure to find activity in at least onecompound meeting the specifications of each proviso.

Preferred compounds of Formula I are compounds selected from the groupconsisting of:

Additional preferred compounds of Formula I are compounds selected fromthe group consisting of:

Additional preferred compounds of Formula I are compounds selected fromthe group consisting of:

Still further preferred compounds are made according to the syntheticmethods outlined in Schemes 1-4 where Y is S and selected from the groupsting of:

EX Compound

-   26    (2,3-Dimethyl-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;-   27    (2-Chloro-3-methyl-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;-   28    (3-Chloro-2-methyl-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;-   29    (2-Bromo-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;-   30    (3-Bromo-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;-   31    (4-Methyl-piperazin-1-yl)-(2-phenyl-6H-thieno[2,3-b]pyrrol-5-yl)-methanone;-   32    [2-(4-Chloro-phenyl)-6H-thieno[2,3-b]pyrrol-5-yl]-(4-methyl-piperazin-1-yl)-methanone;-   33    (3-Bromo-4H-thieno[3,2-b]pyrrol-5-yl)-(3,4-dimethyl-piperazin-1-yl)-methanone;-   34    (3,4-Dimethyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone;-   35    (2-Bromo-3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;-   36    (3-Bromo-2-chloro-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;-   37    (2,3-Dichloro-4H-thieno[3,2-b]pyrrol-5-yl)-(3-methyl-piperazin-1-yl)-methanone;-   38    (4-Methyl-piperazin-1-yl)-(2-phenyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone;    and-   39    (4-Methyl-piperazin-1-yl)-[2-(4-trifluoromethyl-phenyl)-4H-thieno[3,2-b]pyrrol-5-yl]-methanone.

The following terms are defined below, and by their usage throughout thedisclosure.

“Alkyl” includes straight chain and branched hydrocarbons with at leastone hydrogen removed to form a radical group. Alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,1-methylpropyl, pentyl, isopentyl, sec-pentyl, hexyl, heptyl, octyl, andso on. Alkyl does not include cycloalkyl.

“Alkenyl” includes straight chain and branched hydrocarbon radicals asabove with at least one carbon-carbon double bond (sp²). Alkenylsinclude ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (or allyl),isopropenyl (or 1-methylvinyl), but-1-enyl, but-2-enyl, butadienyls,pentenyls, hexa-2,4-dienyl, and so on. Alkenyl does not includecycloalkenyl.

“Alkoxy” includes a straight chain or branched alkyl group with aterminal oxygen linking the alkyl group to the rest of the molecule.Alkoxy includes methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy,pentoxy and so on. “Aminoalkyl”, “thioalkyl”, and “sulfonylalkyl” areanalogous to alkoxy, replacing the terminal oxygen atom of alkoxy with,respectively, NH (or NR), S, and SO₂.

“Cycloalkyl” includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, and so on.

“Halo” includes fluoro, chloro, bromo, and iodo, and preferably fluoroor chloro.

“Patient” or “subject” includes mammals such as humans and animals(dogs, cats, horses, rats, rabbits, mice, non-human primates) in need ofobservation, experiment, treatment or prevention in connection with therelevant disease or condition. Preferably, the patient is a human.

“Composition” includes a product comprising the specified ingredients inthe specified amounts as well as any product that results directly orindirectly from combinations of the specified ingredients in thespecified amounts.

The compounds as described above may be made according to processeswithin the skill of the art and/or that are described in the schemes andexamples that follow. To obtain the various compounds herein, startingmaterials may be employed that carry the ultimately desired substituentsthough the reaction scheme with or without protection as appropriate.Alternatively, it may be necessary to employ, in the place of theultimately desired substituent, a suitable group that may be carriedthrough the reaction scheme and replaced as appropriate with the desiredsubstituent.

Referring to Scheme 1, there are disclosed the following notes andadditions. Various R¹ may be obtained from E1 or C1 by treatment with abase and an appropriate alkylating agent. Where R² is halo, it may beobtained by treatment of E1 and C1 with an appropriate halogenatingagent. Where R² is alkyl, it may be obtained by replacing the aldehydeof A1 with a ketone. P may be an alkyl, aryl or benzyl. Suitable basesinclude NaOEt, LDA, NaH, DBU, etc. The conversion of B1 to C1 isthermolytic with typical temperatures ranging from 80 to 200° C.Suitable solvents for the conversion of B1 to C1 are xylene, cumene,diphenylether, etc. Acidic or basic hydrolysis will providedeprotection. In the case where P is benzyl, hydrogenolysis is alsouseful for deprotection. Typical coupling reagents for the conversion ofD1 to E1 include EDCI, HBTU, etc. Typical chlorination agents for theconversion of D1 to E1 include oxalyl chloride and thionyl chloride. Xis a halogenating agent such as Cl₂, N-bromosuccinimide, TAS-F, Br₂,N-chlorosuccinimide, etc.

Referring to Scheme 2, there are disclosed the following notes andadditions. Various R¹ may be obtained from E2 or C2 by treatment with abase and an appropriate alkylating agent. Where R² is halo, it may beobtained by treatment of E2 and C2 with an appropriate halogenatingagent. Where R² is alkyl, it may be obtained by replacing the aldehydeof A2 with a ketone. P may be an alkyl, aryl or benzyl. Suitable basesinclude NaOEt, LDA, NaH, DBU, etc. The conversion of B2 to C2 isthermolytic with typical temperatures ranging from 80 to 200° C.Suitable solvents for the conversion of B2 to C2 are xylene, cumene,diphenylether, etc. Acidic or basic hydrolysis will providedeprotection. In the case where P is benzyl, hydrogenolysis is alsouseful for deprotection. Typical coupling reagents for the conversion ofD2 to E2 include EDCI, HBTU, etc. Typical chlorination agents for theconversion of D2 to E2 include oxalyl chloride and thionyl chloride. Xis a halogenating agent such as Cl₂, N-bromosuccinimide, TAS-F, Br₂,N-chlorosuccinimide, etc.

Referring to Scheme 3, there are disclosed the following notes andadditions. X is a halogenating agent such as Cl₂, N-bromosuccinimide,TAS-F, Br₂, N-chlorosuccinimide, etc.

Referring to Scheme 4, there are disclosed the following notes andadditions. Typical bases include n-BuLi, LDA, t-BuLi, KHMDS.

The expression of the H₄ receptor in immune cells, including someleukocytes and mast cells, establishes it as an important target fortherapeutic intervention in a range of immunological and inflammatorydisorders (such as allergic, chronic, or acute inflammation).Specifically H₄ receptor ligands are expected to be useful for thetreatment or prevention of various mammalian disease states.

Thus, according to the invention, the disclosed compounds, whereantagonists of the H₄ receptor, and compositions are useful for theamelioration of symptoms associated with, the treatment of, and theprevention of, the following conditions and diseases: inflammatorydisorders, asthma, psoriasis, rheumatoid arthritis, ulcerative colitis,Crohn's disease, inflammatory bowel disease, multiple sclerosis,allergic disorders, allergic rhinitis, dermatological disorders,autoimmune disease, lymphatic disorders, atherosclerosis, andimmunodeficiency disorders. The disclosed compounds may also be usefulas adjuvants in chemotherapy or in the treatment of itchy skin.

Aspects of the invention include (a) a pharmaceutical compositioncomprising a compound of formula (I), or one or more preferred compoundsas described herein, and a pharmaceutically acceptable carrier; (b) apackaged drug comprising (1) a pharmaceutical composition comprising acompound of formula (I) and a pharmaceutically acceptable carrier, and(2) instructions for the administration of said composition for thetreatment or prevention of an H₄-mediated disease or condition.

The invention also provides a method for treating an H₄-mediatedcondition in a patient, said method comprising administering to thepatient a pharmaceutically effective amount of a composition comprisinga compound of formula (I) and other disclosed or preferred compounds.For example, the invention features a method for treating an H₄ mediatedcondition in a patient, said method comprising administering to thepatient a pharmaceutically effective H₄-antagonizing amount of acomposition comprising a compound of formula (I).

The effect of an antagonist may also be produced by an inverse agonist.Inverse agonism describes the property of a compound to actively turnoff a receptor that displays constitutive activity. Constitutiveactivity can be identified in cells that have been forced toover-express the human H₄ receptor. Constitutive activity can bemeasured by examining cAMP levels or by measuring a reporter genesensitive to cAMP levels after a treatment with a cAMP-stimulating agentsuch as forskolin. Cells that over-express H₄ receptors will displaylower cAMP levels after forskolin treatment than non-expressing cells.Compounds that behave as H₄ agonists will dose-dependently lowerforskolin-stimulated cAMP levels in H₄-expressing cells. Compounds thatbehave as inverse H₄ agonists will dose-dependently stimulate cAMPlevels in H₄-expressing cells. Compounds that behave as H₄ antagonistswill block either H₄ agonist-induced inhibition of cAMP or inverse H₄agonist-induced increases in cAMP.

Further embodiments of the invention include disclosed compounds thatare inhibitors of a mammalian histamine H₄ receptor function, inhibitorsof inflammation or inflammatory responses in vivo or in vitro,modulators of the expression of a mammalian histamine H₄ receptorprotein, inhibitors of polymorphonuclear leukocyte activation in vivo orin vitro, or combinations of the above, and corresponding methods oftreatment, prophylaxis, and diagnosis comprising the use of a disclosedcompound.

Those skilled in the art will be able to determine, according to knownmethods, the appropriate dosage for a patient, taking into accountfactors such as age, weight, general health, the type of symptomsrequiring treatment, and the presence of other medications. In general,an effective amount will be between 0.01 and 1000 mg/kg per day,preferably between 0.5 and 300 mg/kg body weight, and daily dosages willbe between 10 and 5000 mg for an adult subject of normal weight.Capsules, tablets or other formulations (such as liquids and film-coatedtablets) may be of between 0.5 and 200 mg, such as 1, 3, 5, 10, 15, 25,35, 50 mg, 60 mg, and 100 mg and can be administered according to thedisclosed methods.

Dosage unit forms include tablets, capsules, pills, powders, granules,aqueous and nonaqueous oral solutions and suspensions, and parenteralsolutions packaged in containers adapted for subdivision into individualdoses. Dosage unit forms can also be adapted for various methods ofadministration, including controlled release formulations, such assubcutaneous implants. Administration methods include oral, rectal,parenteral (intravenous, intramuscular, subcutaneous), intracisternal,intravaginal, intraperitoneal, intravesical, local (drops, powders,ointments, gels or cream), and by inhalation (a buccal or nasal spray).

Parenteral formulations include pharmaceutically acceptable aqueous ornonaqueous solutions, dispersion, suspensions, emulsions, and sterilepowders for the preparation thereof. Examples of carriers include water,ethanol, polyols (propylene glycol, polyethylene glycol), vegetableoils, and injectable organic esters such as ethyl oleate. Fluidity canbe maintained by the use of a coating such as lecithin, a surfactant, ormaintaining appropriate particle size. Carriers for solid dosage formsinclude (a) fillers or extenders, (b) binders, (c) humectants, (d)disintegrating agents, (e) solution retarders, (f) absorptionaccelerators, (g) adsorbants, (h) lubricants, (i) buffering agents, and(k) propellants.

Compositions may also contain adjuvants such as preserving, wetting,emulsifying, and dispensing agents; antimicrobial agents such asparabens, chlorobutanol, phenol, and sorbic acid; isotonic agents suchas a sugar or sodium chloride; absorption-prolonging agents such asaluminum monostearate and gelatin; and absorption-enhancing agents.

EXAMPLES

General Synthetic Procedures

Procedure A: Annulation of Aldehyde with Ethyl Azidoacetate

A solution of aldehyde A1, A2 or A3 (1 equiv) and ethyl azidoacetate (4equiv) was added dropwise to a solution of NaOEt (4 equiv) in EtOH (0.15M) at 0° C. The reaction mixture was stirred at 0° C. for 1 h and atroom temperature for an additional 1 h. The reaction mixture was thenpoured into satd aq NH₄Cl and extracted with ether. The combinedorganics were dried (Na₂SO₄) and concentrated in vacuo. The residue waspurified by silica gel column chromatography to provide the desiredacrylate.

A solution of the resultant acrylate in xylene (0.2 M) was heated at145° C. for 10-60 min and then allowed to cool to room temperature. Thexylene solution was either cooled further to induce productcrystallization or directly subjected to silica gel columnchromatography to obtain the desired annulation product.

Procedure B: Ester Hydrolysis

A solution (0.2 M) of the ethyl ester (1 equiv, from Procedure A) andLiOH (5 equiv) in THF/MeOH/H₂O (3:1:1) was heated at 65° C. overnight,cooled to room temperature, acidified with 2 N HCl, and extracted withEtOAc. The organic layer was separated, dried over Na₂SO₄, andconcentrated to give the desired crude acid, which was taken to the nextstep without further purification.

Procedure C: Amide Formation Using1-(3-Dimethylaminopropyl)-3-ethylcarbodimide Hydrochloride (EDCI)

A mixture of acid (1 equiv, from Procedure B), amine (1.5 equiv) andEDCI (2.0 equiv) in CH₂Cl₂ (0.2 M) was stirred at room temperatureovernight and then partitioned between CH₂Cl₂ and satd aq NaHCO₃. Theorganic layer was separated, washed with H₂O, dried over Na₂SO₄, andconcentrated. The crude product was further purified by silica gelcolumn chromatography.

Procedure D: Amide Formation via Acyl Chloride Intermediate

A mixture of acid (1 equiv, from Procedure B) in CH₂Cl₂ (0.5 M) wastreated at 0° C. with oxalyl chloride (1.2 equiv) followed by 1-2 dropsof DMF. The reaction mixture was stirred at 0° C. for 30 min then slowlywarmed to room temperature and stirred for an additional 1 h. Allvolatiles were removed to provide the crude acyl chloride.

The resultant acyl chloride was treated with amine (5.0 equiv) in CH₂Cl₂(0.2 M) and allowed to stir at room temperature for 3 h. The reactionmixture was partitioned between CH₂Cl₂ and satd aq NaHCO₃. The organiclayer was separated, washed with H₂O, dried over Na₂SO₄, andconcentrated. The crude product was further purified with silica gelcolumn chromatography.

General Analytical Procedures

NMR spectra were obtained on either a Bruker model DPX400 (400 MHz) orDPX500 (500 MHz) spectrometer. The format of the 1H NMR data below is:chemical shift in ppm down field of the tetramethylsilane reference(multiplicity, coupling constant J in Hz, integration).

Mass spectra were obtained on a Hewlett Packard (Agilent) series 1100MSD using electrospray ionization (ESI) in either positive or negativemode as indicated. The “mass calculated” for a molecular formula is themonoisotopic mass of the compound.

Silica Gel Column Chromatography:

Normal-phase column chromatography was accomplished using an ISCO Foxy200 system employing one of the following commercially availableprepacked columns: ISCO Redisep (SiO₂, 10 g, 12 g, 35 g, 40 g, or 120g).

Example 1

(4-Methyl-piperazin-1-yl)-(6H-thieno[2,3-b]pyrrol-5-yl)-methanone

A. 6H-Thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester.Thiophene-3-carbaldehyde (2.24 g, 20 mmol) was annulated according toProcedure A to provide the title compound (1.2 g, 31%) as a white solid.TLC (silica, 20% EtOAc/hexanes): R_(f)=0.50. ¹H NMR (CDCl₃, 400 MHz):10.30 (br s, 1H), 7.10 (d, J=1.9 Hz, 1H), 6.96 (d, J=5.4 Hz, 1H), 6.87(d, J=5.4 Hz, 1H), 4.39 (q, J=7.1 Hz, 2H), 1.38 (t, J=7.1 Hz, 3H).

B. 6H-Thieno[2,3-b]pyrrole-5-carboxylic acid.6H-Thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (835 mg, 4.3 mmol)was hydrolyzed according to Procedure B to provide the crude acid as apale-yellow solid. ¹H NMR (CD₃OD, 400 MHz): 7.02 (s, 1H), 6.96 (s, 1H),6.95 (s, 1H).

C. (4-Methyl-piperazin-1-yl)-(6H-thieno[2,3-b]pyrrol-5-yl)-methanone.6H-Thieno[2,3-b]pyrrole-5-carboxylic acid (60 mg, 0.35 mmol) was coupledwith N-methylpiperazine according to Procedure C to provide the titlecompound (44 mg, 50%) as a light yellow solid. TLC (silica, 10%MeOH/CH₂Cl₂): R_(f)=0.4. MS (electrospray): exact mass calculated forC₁₂H₁₅N₃OS, 249.09; m/z found, 250.1 [M+H]⁺. ¹H NMR (CD₃OD, 400 MHz, TFAsalt): 6.97 (s, 1H), 6.96 (s, 1H), 6.85 (s, 1H), 4.20-3.10 (m, 8H), 2.96(s, 3H).

Example 2

(Hexahydro-pyrrolo[1,2-a]pyrazin-2-yl)-(6H-thieno[2,3-b]pyrrol-5-yl)-methanone

6H-Thieno[2,3-b]pyrrole-5-carboxylic acid (60 mg, 0.35 mmol) was coupledwith octahydro-pyrrolo[1,2-a]pyrazine according to Procedure C toprovide the title compound (34 mg, 35%) as a light yellow solid. TLC(silica, 10% MeOH/CH₂Cl₂): R_(f)=0.4. MS (electrospray): exact masscalculated for C₁₄H₁₇N₃OS, 275.11; m/z found, 276.2 [M+H]⁺. ¹H NMR(CDCl₃, 400 MHz): 11.1 (br s, 1H), 6.96 (d, J=5.4 Hz, 1H), 6.87 (d,J=5.4 Hz, 1H), 6.71 (d, J=1.9 Hz, 1H), 4.84 (d, J=12.2 Hz, 1H), 4.70 (d,J=12.2 Hz, 1H), 3.30-2.90 (m, 4H), 2.30-1.40 (m, 7H).

Example 3

(2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

A. 2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester. Asolution of 6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (580mg, 3.0 mmol) in acetic acid (6 mL) and CHCl₃ (6 mL) was treated withthree portions of N-chlorosuccinimide (total 415 mg, 3.15 mmol) at 0° C.over 2 h. The reaction mixture was slowly warmed to room temperature andstirred overnight. The CHCl₃ was then removed, and the residue wasbasified with 4 N NaOH and extracted with EtOAc. The combined organicswere washed with satd aq NaHCO₃, dried over Na₂SO₄, and concentrated.Column chromatography (SiO₂, 5-10% EtOAc/hexanes) gave 600 mg (88%) of awhite solid. TLC (silica, 20% EtOAc/hexanes): R_(f)=0.5. ¹H NMR (CDCl₃,400 MHz): 10.5 (br s, 1H), 6.97 (d, J=2.0 Hz, 1H), 6.85 (s, 1H), 4.39(q, J=7.2 Hz, 2H), 1.35 (t, J=7.2 Hz, 3H).

B.(2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.

2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (102 mg,0.45 mmol) was hydrolyzed (Procedure B) and coupled withN-methylpiperazine (procedure D) to provide the title compound (102 mg,80% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.4. MS (electrospray): exact mass calculated for C₁₂H₁₄ClN₃OS,283.05; m/z found, 284.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.5 (br s,1H), 6.87 (s, 1H), 6.61 (d, J=1.8 Hz, 1H), 3.92 (t, J=5.1 Hz, 4H), 2.50(t, J=5.1 Hz, 4H), 2.35 (s, 3H).

Example 4

(2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-(hexahydro-pyrrolo[1,2-a]pyrazin-2-yl)-methanone

2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (102 mg,0.45 mmol) was hydrolyzed (Procedure B) and then coupled withoctahydro-pyrrolo[1,2-a]pyrazine (Procedure D) to provide the titlecompound (108 mg, 78% for two steps) as an off-white solid. TLC (silica,10% MeOH/CH₂Cl₂): R_(f)=0.35. MS (electrospray): exact mass calculatedfor C₁₄H₁₆ClN₃OS, 309.07; m/z found, 310.1 [M+H]⁺. ¹H NMR (CDCl₃, 400MHz): 11.1 (br s, 1H), 6.86 (s, 1H), 6.62 (s, 1H), 4.79 (d, J=11.8 Hz,1H), 4.67 (d, J=11.8 Hz, 1H), 3.30-2.90 (m, 4H), 2.30-1.40 (m, 7H).

Example 5

(2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-piperazin-1-yl-methanone

2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (102 mg,0.45 mmol) was hydrolyzed (Procedure B) and then coupled with piperazine(Procedure D) to provide the title compound (42 mg, 35% for two steps)as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂): R_(f)=0.15. MS(electrospray): exact mass calculated for C₁₁H₁₂ClN₃OS, 269.04; m/zfound, 270.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.5 (br s, 1H), 6.87 (s,1H), 6.61 (s, 1H), 3.87 (t, J=4.8 Hz, 4H), 2.96 (t, J=5.2 Hz, 4H).

Example 6

(4H-Furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

A. 4H-Furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester.Furan-2-carbaldehyde (1.92 g, 20 mmol) was annulated according toprocedure A to provide the title compound (1.97 g, 55%) as a whitesolid. TLC (silica, 20% EtOAc/hexanes): R_(f)=0.50. ¹H NMR (CDCl₃, 400MHz): 8.95 (br s, 1H), 7.51 (d, J=2.2 Hz, 1H), 6.81-6.80 (m, 1H),6.46-6.45 (m, 1H), 4.35 (q, J=7.1 Hz, 2H), 1.38 (t, J=7.1 Hz, 3H).

B. (4H-Furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.4H-Furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester (200 mg, 1.12 mmol)was hydrolyzed (Procedure B) and coupled with N-methylpiperazine(Procedure D) to provide the title compound (185 mg, 71% for two steps)as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂): R_(f)=0.4. MS(electrospray): exact mass calculated for C₁₂H₁₅N₃O₂, 233.12; m/z found,234.2 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.3 (br s, 1H), 7.43 (d, J=2.2Hz, 1H), 6.43-6.42 (m, 2H), 3.90 (t, J=5.0 Hz, 4H), 2.47 (t, J=5.1 Hz,4H), 2.32 (s, 3H).

Example 7

(4-Methyl-piperazin-1-yl)-(4H-thieno[3,2-b]pyrrol-5-yl)-methanone

A. 4H-Thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester. To a solutionof thiophene-2-carbaldehyde (1.10 mL, 11.7 mmol) and ethyl azidoacetate(1.4 mL, 11.7 mmol) in EtOH (35 mL) cooled to 0° C. was added NaOEt (1.0g, 14.7 mmol) in one portion. The mixture was allowed to reach roomtemperature over 14 h and was then poured into water (400 mL) andextracted with CH₂Cl₂ (3×50 mL). The combined organics were washed withwater and brine, dried over Na₂SO₄, and concentrated. The residue wastaken up in xylenes (10 mL), and the resulting solution was refluxed for1 h. The solution was cooled and then loaded directly onto silica geland purified (35 g SiO₂, 10-20% EtOAc/hexanes) to reveal 0.12 g (5%) ofa yellowish solid. ¹H NMR (400 MHz, CDCl₃): 9.06 (br s, 1H), 7.33 (d,J=5.3 Hz, 1H), 7.15-7.14 (m, 1H), 6.96 (dd, J=5.3, 0.8 Hz, 1H), 4.37 (q,J=7.3 Hz, 2H), 1.39 (t, J=7.3 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): 161.3,140.9, 129.2, 126.9, 124.6, 110.9, 107.3, 60.4, 14.2.

B. (4-Methyl-piperazin-1-yl)-(4H-thieno[3,2-b]pyrrol-5-yl)-methanone. Toa solution of 4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester(98.5 mg, 0.50 mmol) in wet THF (3 mL) was added LiOH (129 mg, 3 mmol).This mixture was stirred at room temperature for 3 days. The reactionmixture was diluted with water (50 mL), and 1 M HCL was added to adjustthe pH to about 3. This mixture was then extracted with EtOAc, and thecombined organics were dried over Na₂SO₄. The solvent was removed toreveal 73.4 mg (87%) of the free acid, which was used in the couplingevent without further purification. The acid (73.4 mg, 0.44 mmol) wastaken up in THF (3 mL), and CDI (87.1 mg, 0.54 mmol) was added in oneportion. The reaction mixture was stirred for 1 h. To this mixture wasthen added 1-methylpiperazine (70 μL), and the mixture stirred for anadditional 6 h. The reaction mixture was diluted with EtOAc, washed withwater, NaHCO₃ (aq) and then brine, and subsequently purified by columnchromatography (10 g SiO₂, 1-8% MeOH (2 M NH₃)/CH₂Cl₂) to reveal 55.6 mg(51%) of the title compound. ¹H NMR (400 MHz, CDCl₃): 9.26 (br s, 1H),7.26 (d, J=5.3 Hz, 1H), 6.97 (dd, J=5.3, 0.8 Hz, 1H), 6.75-6.74 (m, 1H),4.07-3.88 (m, 4H), 2.68-2.48 (m, 4H), 2.43 (br s, 3H). MS(electrospray): exact mass calculated for C₁₂H₁₅N₃OS, 249.09; m/z found,250.1 [M+H]⁺.

Example 8

Piperazin-1-yl-(4H-thieno[3,2-b]pyrrol-5-yl)-methanone

4H-Thieno[3,2-b]pyrrole-5-carboxylic acid (50 mg, 0.30 mmol) was coupledwith piperazine according to Procedure D to provide the title compound(25 mg, 35%) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.15. MS (electrospray): exact mass calculated for C₁₁H₁₃N₃OS,235.08; m/z found, 236.1 [M+H]⁺. ¹H NMR (CD₃OD, 400 MHz): 7.33 (d, J=5.3Hz, 1H), 6.98 (dd, J=5.2, 0.7 Hz, 1H), 6.89 (d, J=0.6 Hz, 1H), 4.08 (t,J=5.3 Hz, 4H), 3.50-3.20 (m, 4H).

Example 9

(3-Methyl-piperazin-1-yl)-(4H-thieno[3,2-b]pyrrol-5-yl)-methanone

4H-Thieno[3,2-b]pyrrole-5-carboxylic acid (50 mg, 0.30 mmol) was coupledwith 2-methylpiperazine according to Procedure D to provide the titlecompound (58 mg, 78%) as an off-white solid. TLC (silica, 10%MeOH/CH₂Cl₂): R_(f)=0.15. MS (electrospray): exact mass calculated forC₁₂H₁₅N₃OS, 249.09; m/z found, 250.1 [M+H]⁺. ¹H NMR (CD₃OD, 400 MHz):7.33 (d, J=5.3 Hz, 1H), 6.98 (d, J=5.3 Hz, 1H), 6.88 (s, 1H), 4.62-4.56(m, 2H), 3.50-3.20 (m, 5H), 1.36 (d, J=6.6 Hz, 3H).

Example 10

(2-Chloro-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

A. 2-Chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester.5-Chloro-thiophene-2-carbaldehyde (2.92 g, 20 mmol) was annulatedaccording to Procedure A to provide the title compound (2.8 g, 61%) as awhite solid. TLC (silica, 20% EtOAc/hexanes): R_(f)=0.48. ¹H NMR (CDCl₃,400 MHz): 9.10 (br s, 1H), 7.04 (dd, J=1.9, 0.7 Hz, 1H), 6.89 (d, J=0.7Hz, 1H), 4.37 (q, J=7.2 Hz, 2H), 1.39 (t, J=7.2 Hz, 3H).

B.(2-Chloro-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.

2-Chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (230 mg,1.0 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure C) to provide the title compound (128 mg,45% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.4. MS (electrospray): exact mass calculated for C₁₂H₁₄ClN₃OS,283.05; m/z found, 284.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.1 (br s,1H), 6.88 (s, 1H), 6.64 (d, J=1.4 Hz, 1H), 3.91 (t, J=4.4 Hz, 4H), 2.49(t, J=5.1 Hz, 4H), 2.35 (s, 3H).

Example 11

(2-Chloro-4H-thieno[3,2-b]pyrrol-5-yl)-(hexahydro-pyrrolo[1,2-a]pyrazin-2-yl)-methanone

2-Chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (230 mg,1.0 mmol) was hydrolyzed (Procedure B) and then coupled withoctahydro-pyrrolo[1,2-a]pyrazine (Procedure C) to provide the titlecompound (93 mg, 30% for two steps) as an off-white solid. TLC (silica,10% MeOH/CH₂Cl₂): R_(f)=0.4. MS (electrospray): exact mass calculatedfor C₁₄H₁₆ClN₃OS, 309.07; m/z found, 310.1 [M+H]⁺. ¹H NMR (CDCl₃, 400MHz): 10.9 (br s, 1H), 6.86 (s, 1H), 6.64 (d, J=1.4 Hz, 1H), 4.77 (d,J=12.2 Hz, 1H), 4.65 (d, J=12.7 Hz, 1H), 3.30-2.90 (m, 4H), 2.30-1.40(m, 7H).

Example 12

(3-Bromo-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.

A. 3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester.4-Bromo-thiophene-2-carbaldehyde (3.8 g, 20 mmol) was annulatedaccording to Procedure A to provide the title compound (1.2 g, 22%) as awhite solid. TLC (silica, 20% EtOAc/hexanes): R_(f)=0.48. ¹H NMR (CDCl₃,400 MHz): 9.58 (br s, 1H), 7.20 (s, 1H), 7.15 (d, J=1.5 Hz, 1H), 4.41(q, J=7.2 Hz, 2H), 1.39 (t, J=7.2 Hz, 3H).

B.(3-Bromo-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.

3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (67 mg,0.24 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (65 mg,82% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.4. MS (electrospray): exact mass calculated for C₁₂H₁₄BrN₃OS,327.00; m/z found, 328.0 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 9.95 (br s,1H), 7.11 (s, 1H), 6.73 (d, J=1.8 Hz, 1H), 3.91 (t, J=5.1 Hz, 4H), 2.49(t, J=5.1 Hz, 4H), 2.34 (s, 3H).

Example 13

(4-Methyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone

A. 4-methyl-thiophene-2-carbaldehyde. A solution of 3-methylthiophene(6.76 mL, 70 mmol) in ether (70 mL) was treated with n-butyllithium (2.5M in hexanes, 28.6 mL, 71.4 mmol) at such a rate that a slight refluxwas maintained. The reaction mixture was heated to reflux for 15 min andthen DMF (7.0 mL, 91 mmol) in ether (30 mL) was added. After stirringfor 4 h, the reaction was quenched with addition of satd aq NH₄Cl (200mL). The organic layer was separated, washed with brine and then H₂O,dried over Na₂SO₄, and concentrated. Column chromatography (SiO₂, 5-10%EtOAc/hexanes) provided a mixture of 4-methyl-thiophene-2-carbaldehydeand 3-methyl-thiophene-2-carbaldehyde (4.4:1, 8.1 g, 92%) as a lightyellow oil. TLC (silica, 10% EtOAc/hexanes): R_(f)=0.55. For4-methyl-thiophene-2-carbaldehyde: ¹H NMR (CDCl₃, 400 MHz): 9.95 (s,1H), 7.58 (d, J=1.2 Hz, 1H), 7.37-7.35 (m, 1H), 2.32 (s, 3H). For3-methyl-thiophene-2-carbaldehyde: ¹H NMR (CDCl₃, 400 MHz): 10.02 (s,1H), 7.64 (d, J=4.6 Hz, 1H), 6.97 (d, J=4.6 Hz, 1H), 2.58 (s, 3H).

B. 3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester. Themixture of 4-methyl-thiophene-2-carbaldehyde and3-methyl-thiophene-2-carbaldehyde (2.84 g, 22.5 mmol) was annulatedaccording to Procedure A to provide the title compound (2.5 g, 65%) as awhite solid. TLC (silica, 20% EtOAc/hexanes): R_(f)=0.45. ¹H NMR (CDCl₃,400 MHz): 9.95 (br s, 1H), 7.12 (d, J=1.9 Hz, 1H), 6.90 (d, J=1.2 Hz,1H), 4.39 (q, J=7.2 Hz, 2H), 2.35 (s, 3H), 1.39 (t, J=7.2 Hz, 3H).

C.(4-Methyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone.3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (200 mg,0.96 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (197 mg,78% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.4. MS (electrospray): exact mass calculated for C₁₃H₁₇N₃OS,263.11; m/z found, 264.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 11.10 (br s,1H), 6.76 (d, J=1.2 Hz, 1H), 6.69 (d, J=2.0 Hz, 1H), 3.94-3.90 (m, 4H),2.47 (t, J=5.1 Hz, 4H), 2.33 (s, 3H), 2.25 (s, 3H).

Example 14

(2-Methyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

A. 2-Methyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester.5-Methyl-furan-2-carbaldehyde (2.2 g, 20 mmol) was annulated accordingto Procedure A to provide the title compound (2.89 g, 75%) as a whitesolid. TLC (silica, 10% EtOAc/hexanes): R_(f)=0.4. ¹H NMR (CDCl₃, 400MHz): 9.50 (br s, 1H), 6.73 (s, 1H), 6.04 (s, 1H), 4.35 (q, J=7.2 Hz,2H), 2.37 (s, 3H), 1.35 (t, J=7.2 Hz, 3H).

B.(2-Methyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.2-Methyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester (200 mg,1.04 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (208 mg,81% for two steps) as a white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.35. MS (electrospray): exact mass calculated for C₁₃H₁₇N₃O₂,247.13; m/z found, 248.2 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 9.85 (br s,1H), 6.36 (s, 1H), 6.07 (s, 1H), 3.87 (t, J=5.0 Hz, 4H), 2.47 (t, J=5.2Hz, 4H), 2.39 (s, 3H), 2.33 (s, 3H).

Example 15

(2,3-Dimethyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

A. 2,3-Dimethyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester.4,5-Dimethyl-furan-2-carbaldehyde (2.2 g, 18 mmol) was annulatedaccording to Procedure A to provide the title compound (1.76 g, 48%) asan off-white solid. TLC (silica, 10% EtOAc/hexanes): R_(f)=0.35. ¹H NMR(CDCl₃, 400 MHz): 8.95 (br s, 1H), 6.69 (d, J=1.7 Hz, 1H), 4.32 (q,J=7.2 Hz, 2H), 2.33 (s, 3H), 2.08 (s, 3H), 1.37 (t, J=7.2 Hz, 3H).

B.(2,3-Dimethyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.2,3-Dimethyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester (200mg, 0.97 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (190 mg,75% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.35. MS (electrospray): exact mass calculated for C₁₄H₁₉N₃O₂,261.15; m/z found, 261.8 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 9.95 (br s,1H), 6.32 (d, J=1.8 Hz, 1H), 3.88 (t, J=5.0 Hz, 4H), 2.47 (t, J=5.1 Hz,4H), 2.33 (s, 3H), 2.31 (s, 3H), 2.06 (s, 3H).

Example 16

(2,3-Dimethyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

A. 2,3-Dimethyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester.4,5-Dimethyl-thiophene-2-carbaldehyde (2.0 g, 14 mmol) was annulatedaccording to Procedure A to provide the title compound (160 mg, 5%) as awhite solid. TLC (silica, 10% EtOAc/hexanes): R_(f)=0.40. ¹H NMR (CDCl₃,400 MHz): 9.50 (br s, 1H), 7.05 (d, J=1.9 Hz, 1H), 4.36 (q, J=7.2 Hz,2H), 2.41 (s, 3H), 2.22), (s, 3H), 1.38 (t, J=7.2 Hz, 3H).

B.(2,3-Dimethyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.2,3-Dimethyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (68mg, 0.30 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (67 mg,80% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.35. MS (electrospray): exact mass calculated for C₁₄H₁₉N₃OS,277.12; m/z found, 278.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.95 (br s,1H), 6.63 (d, J=1.9 Hz, 1H), 3.92 (t, J=4.5 Hz, 4H), 2.46 (t, J=5.0 Hz,4H), 2.36 (s, 3H), 2.33 (s, 3H), 2.13 (s, 3H).

Examples 17-25

The following compounds were made according to the synthetic methodsoutlined in Schemes 1-4:

EX Compound

-   17    (2,3-Dichloro-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;-   18 (2-Methyl-4H-furo[3,2-b]pyrrol-5-yl)-piperazin-1-yl-methanone;-   19 (3-Bromo-4H-thieno[3,2-b]pyrrol-5-yl)-piperazin-1-yl-methanone;-   20    (3-Bromo-4H-thieno[3,2-b]pyrrol-5-yl)-(3-methyl-piperazin-1-yl)-methanone;-   21 (3-Methyl-4H-thieno[3,2-b]pyrrol-5-yl)-piperazin-1-yl-methanone;-   22    (3-Methyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone;-   23    (2-Chloro-3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;-   24    (2-Chloro-3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-piperazin-1-yl-methanone;    and-   25    (2,3-Dichloro-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.

Biological Examples

Binding Assay on Recombinant Human Histamine H₄ Receptor

SK-N-MC cells or COS7 cells were transiently transfected with pH4R andgrown in 150 cm² tissue culture dishes. Cells were washed with salinesolution, scraped with a cell scraper and collected by centrifugation(1000 rpm, 5 min). Cell membranes were prepared by homogenization of thecell pellet in 20 mM Tris-HCl with a polytron tissue homogenizer for 10s at high speed. Homogenate was centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was then collected and centrifuged at 20,000×g for 25min at 4° C. The final pellet was resuspended in 50 mM Tris-HCl. Cellmembranes were incubated with ³H-histamine (5-70 nM) in the presence orabsence of excess histamine (10000 nM). Incubation occurred at roomtemperature for 45 min. Membranes were harvested by rapid filtrationover Whatman GF/C filters and washed 4 times with ice-cold 50 mM TrisHCl. Filters were then dried, mixed with scintillant and counted forradioactivity. SK-N-MC or COS7 cells expressing human histamine H₄receptor were used to measure the affinity of binding of other compoundsand their ability to displace ³H-ligand binding by incubating theabove-described reaction in the presence of various concentrations ofinhibitor or compound to be tested. For competition binding studiesusing ³H-histamine, K_(i) values were calculated, based on anexperimentally determined K_(D) value of 5 nM and a ligand concentrationof 5 nM, according to Y.-C. Cheng and W. H. Prusoff (Biochem. Pharmacol.1973, 22(23):3099-3108): K_(i)=(IC₅₀)/(1+([L]/(K_(D))). BINDING ASSAYRESULTS EX K_(i) (nM) 1 85 2 461 3 25 4 176 5 56 6 840 7 125 8 343 9 73310 40 11 715 12 56 13 21 14 343 15 140 16 5 17 10 18 770 19 410 20 98021 80 22 161 23 3 24 30 25 5.5Mast Cell Chemotaxis Assay

Mast cell accumulation in mucosal epithelia is a well-knowncharacteristic of allergic rhinitis and asthma. Transwells (Costar,Cambridge, Mass.) of a pore size 8 μm were coated with 100 μL of 100ng/mL human fibronectin (Sigma) for 2 h at room temperature. Afterremoval of the fibronectin, 600 μL of RPMI with 5% BSA, in the presenceof 10 μM histamine, was added to the bottom chamber. To test the varioushistamine receptor (HR) antagonists, 10 μM and/or 1 μM Solutions of thetest compounds were added to the top and bottom chambers. Mast cells(2×10⁵/well) were added to the top chamber. The plates were Incubatedfor 3 h at 37° C. Transwells were removed and the cells in the Bottomchamber were counted for sixty seconds using a flow cytometer. 10 μMHistamine HR Antagonist (μM): Binding 10 1 Assay EX % Inh Stdev % InhStdev K_(i) (nM) 3 106 4 103 0 25 4 <5 — <5 — 176 10 92 3 40 13 60 20 21 20 <5 — <5 — 980Cell-Type Distribution of H₄ Expression

RNA was prepared from the different cells using an RNeasy kit (Qiagen,Valencia, Calif.) according to the manufacturer's instructions. RNAsamples (5 μg) were run on an RNA gel and then transferred overnight toa nylon blot (Hybond, Amersham Pharmacia Biotech, Piscataway, N.J.). Theblot was pre-hybridized with ExpressHyb solution (CLONTECH) for 30 minat 68° C. The H₄ receptor DNA was labeled using the Rediprime II kit(Amersham Pharmacia Biotech). The blot was hybridized for 2 h at 68° C.,followed by one wash step (23 SSC and 0.05% SDS) of 40 min at roomtemperature, and a second wash step (0.13 SSC and 0.1% SDS) of 40 min at50° C. The blot was exposed to X-ray film at −70° C. with twointensifying screens overnight.

Results

The Northern Blot results indicate that the H₄ receptor is expressed onbone marrow-derived mast cells (BMMC), peritoneal mast cells, andeosinophils. These positive results are consistent with the publishedliterature (e.g. Oda et al., Nguyen et al., and Morse et al. in theBackground section). However, the negative results of the Northern Blotexperiment, such as the finding of apparently no measurable levels of H₄receptor expressed by neutrophils, differ somewhat from the aboveliterature findings. This may be explained by the differentmethodologies used. Accumulation of mast cells and eosinophils inaffected tissues is one of the principal characteristics of allergicrhinitis and asthma. Since H₄ receptor expression is limited to thesecell types; H₄ receptor signalling is likely to mediate the infiltrationof mast cells and eosinophils in response to histamine. Additionalinvestigation may also clarify these issues. The following table reportsthe Cell-type Distribution of H₄ Expression by Northern Blot. SpeciesCell Type H₄ Human Eosinophils + Immature Dendritic Cells − MatureDendritic Cells − CD14⁺ Monocytes − CD4⁺ T Cells − CD8⁺ T Cells − BCells − Neutrophils − Mouse/(Rat) Eosinophils + Peritoneal Mast Cells(Rat) + BMMC + BM Derived Macrophages − Peritoneal Macrophages − CD4⁺ TCells − B Cells −The Inhibition of Eosinophil Shape Change by Histamine H₄ ReceptorAntagonists

Eosinophil accumulation in sites of allergic reaction is a well-knowncharacteristic of allergic rhinitis and asthma. This exampledemonstrates that histamine H₄ receptor antagonists can block the shapechange response in human eosinophils in response to histamine. Shapechange is a cellular characteristic that precedes eosinophil chemotaxis.

Methods

Human granulocytes were isolated from human blood by a Ficoll gradient.The red blood cells were lysed with 5-10× Qiagen lysis buffer at roomtemperature for 5-7 min. Granulocytes were harvested and washed oncewith FACS buffer. The cells were resuspended at a density of 2×10⁶cells/mL in reaction buffer. To test inhibition by specific histaminereceptor antagonists, 90 μL of the cell suspension (˜2×10⁵ cells) wasincubated with 10 μM of one of the various test compound solutions.After 30 min, 11 μL of one of the various concentrations of histaminewas added. Ten minutes later the cells were transferred to ice and fixedwith 250 μL of ice-cold fixative buffer (2% formaldehyde) for 1 min. Theshape change was quantitated using a gated autofluorescence forwardscatter assay (GAFS) (Byran et al., Am. J. Crit. Care Med.2002,165:1602-1609).

Results—Histamine Mediates Eosinophil Shape Change Through H₄ Receptor

The change in shape of eosinophils is due to cytoskeletal changes thatpreceed chemotaxis and thus is a measure of chemotaxis. The data in thefollowing table show that histamine induces a dose-dependent shapechange in eosinophils. Histamine receptor (HR) antagonists were used tosort out which histamine receptor is responsible for the shape change.Antagonists specific for the histamine H₁ receptor (diphenhydramine) orthe H₂ receptor (ranatidine) did not alter the histamine-induced shapechange. However, a dual H₃/H₄ antagonist (thioperamide) and a specifichistamine H₄ receptor antagonist((5-Chloro-1H-indol-2-yl)-(4-methyl-piperazin-1-yl)-methanone, K_(i)=5nM) inhibited histamine-induced eosinophil shape change with an IC₅₀ of1.5 and 0.27 μM, respectively. Histamine Fold Change (μM): 10 1 0.1 0.010 No HR 1.34 1.31 1.21 1.01 1.00 Antagonist 10 μM H₄ 1.09 1.05 1.05 1.011.00 Antagonist 10 μM 1.08 1.05 1.01 1.04 1.00 Thiop 10 μM 1.63 1.501.18 1.03 1.00 Diphen 10 μM 1.64 1.49 1.21 1.04 1.00 RanatThe Inhibition of Eosinophil Chemotaxis by Histamine H₄ ReceptorAntagonists

Eosinophil accumulation in sites of allergic reaction is a well-knowncharacteristic of allergic rhinitis and asthma. Eosinophils are purifiedfrom human blood with standard methods. Chemotaxis assays are carriedout using transwells (Costar, Cambridge, Mass.) of a pore size 5 μmcoated with 100 μL of 100 ng/mL human fibronectin (Sigma) for 2 h atroom temperature. After removal of the fibronectin, 600 μL of RPMI with5% BSA in the presence of histamine (ranging from 1.25-20 μM) is addedto the bottom chamber. To test the various histamine receptorantagonists 10 μM of the test compounds can be added to the top andbottom chambers. Eosinophils will be added to the top chamber whereashistamine or chemotactic factors will be placed in the lower chamber.The plates are incubated for 3 h at 37° C. Transwells are removed andthe number of cells in the bottom chamber can be counted for 60 s usinga flow cytometer, or can be quantitated by using Giemsa staining.

The Inhibition of Zymosan-Induced Peritonitis in Mice by Histamine H₄Receptor Antagonists

It has been demonstrated that histamine H₄ receptor antagonists canblock the peritonitis induced by zymosan, which is the insolublepolysaccharide component on the cell wall of Saccharomyces cerevisiae.This is commonly used to induce peritonitis in mice and appears to actin a mast cell-dependent manner. Compounds of the present invention canbe tested in such a model to demonstrate their use as anti-inflammatoryagents. At time 0 mice are given compound or PBS, either s.c. or p.o.Fifteen minutes later each mouse receives 1 mg zymosan A (Sigma) i.p.The mice are sacrificed 4 h later, and the peritoneal cavities arewashed with 3 mL of PBS containing 3 mM EDTA. The number of migratedleukocytes is determined by taking an aliquot (100 μL) of the lavagefluid and diluting 1:10 in Turk's solution (0.01% crystal violet in 3%acetic acid). The samples are then vortexed, and 10 μL of the stainedcell solution is placed in a Neubauer haemocytometer. Differential cellcounts are performed using a light microscope (Olympus B061). In view oftheir chromatic characteristics and their nucleus and cytoplasmappearance, polymorphonuclear leukocytes (PMN; >95% neutrophils) can beeasily identified. Treatment with zymosan increases the number ofneutrophils, which is representative of an inflammatory response.Treatment with H₄ receptor antagonist will block this increase.

Inhibition of Mast Cell Chemotaxis by H₄ Receptor Antagonist in anAnimal Model of Asthma and Allergic Rhinitis

An animal model will be used to test the observation that mast cellsaccumulate in response to allergic inflammation and that this can beblocked by H₄ receptor antagonists. Compounds of the present inventioncan be tested in this model to demonstrate their use as treatments forallergic rhinitis or asthma. Mice will be sensitized by intraperitonealinjection of ovalbumin/Alum (10 μg in 0.2 ml Al(OH)₃; 2%) on Day 0 andDay 14. On Day 21 through 23 mice will be challenged by PBS orovalbumin, and sacrificed 24 h after the last challenge on Day 24. Asection of the trachea will be removed and fixed in formalin. Paraffinembedding and longitudinal sectioning of tracheas will be performedfollowed by staining of mast cells with toluidine blue. Alternatively,trachea will be frozen in OCT for frozen sectioning, and mast cells willbe identified by IgE staining. Mast cells will be quantified assub-mucosal or sub-epithelial depending on their location within eachtracheal section. Exposure to allergen should increase the number ofsub-epithelial mast cells, and this effect will be blocked by H₄receptor antagonists.

The features and advantages of the invention are apparent to one ofordinary skill in the art. Based on this disclosure, including thesummary, detailed description, background, examples, and claims, one ofordinary skill in the art will be able to make modifications andadaptations to various conditions and usages. Publications describedherein are incorporated by reference in their entirety. These otherembodiments are also within the scope of the invention.

1. A compound of formula (I):

Y is O Z is O or S; n is 1 or 2; m is 1 or 2; n+m is 2 or 3; R¹ is H orC₁₋₆alkyl; R² is H, F, Cl, Br or C₁₋₆alkyl; R³ and R⁴ are,independently, H, C₁₋₄alkyl, C₃₋₆cycloalkyl, C₁₋₄alkyl(C₃₋₆cycloalkyl),cyano, —CF₃, —(CO)NR^(p)R^(q), —(CO)OR^(r), —CH₂NR^(p)R^(q) or—CH₂OR^(r); where R^(p), R^(q) and R^(r) are independently selected fromH, C₁₋₄alkyl, C₃₋₆cycloalkyl, phenyl, —C₁₋₂alkyl(C₃₋₆cycloalkyl), benzylor phenethyl, or R^(p) and R^(q) taken together with the nitrogen towhich they are attached, form a 4-7 membered heterocyclic ring with 0 or1 additional heteroatoms selected from O, S, NH or NC₁₋₆alkyl, and whereany phenyl or alkyl or cycloalkyl moiety of the foregoing is optionallyand independently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; R⁵ and R⁶ are,independently, H or C₁₋₆alkyl; R⁷ is —R^(a), —R^(b)R^(a), —R^(e)—R^(a)or —R^(e)—N(R^(c))(R^(d)), where R^(a) is H, cyano,—(C═O)N(R^(c))(R^(d)), —C(═NH)(NH₂), C₁₋₁₀alkyl, C₂₋₈alkenyl,C₃₋₈cycloalkyl, C₄₋₇heterocyclic radical or phenyl, where theC₄₋₇heterocyclic radical is attached at a carbon atom and contains oneof O, S, NH or NC₁₋₄alkyl, and optionally an additional NH or NC₁₋₆alkylin rings of 5 or 6 or 7 members, where R^(b) is C₁₋₈alkylene orC₂₋₈alkenylene, where R^(e) is C₂₋₈alkylene or C₂₋₈alkenylene, whereR^(c) and R^(d) are each independently H, C₁₋₄alkyl, C₂₋₄alkenyl,C₃₋₆cycloalkyl or phenyl, or R^(c) and R^(d) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₆alkyl, and where any phenyl or alkyl or cycloalkyl moiety of theforegoing is optionally and independently substituted with between 1 and3 substituents selected from C₁₋₃alkyl, halo, hydroxy, amino, andC₁₋₃alkoxy; alternatively, R⁷ may be taken together with an adjacent R⁴as well as their carbon and nitrogen of attachment to form a 5, 6 or 7membered heterocyclic ring, with 0 or 1 additional heteroatoms selectedfrom O, S, NH or NC₁₋₆alkyl, and optionally and independentlysubstituted with between 1 and 3 substituents selected from C₁₋₃alkyl,halo, hydroxy, amino, and C₁₋₃alkoxy; R⁸ and R⁹ are, independently, H,F, Cl, Br, I, C₁₋₄alkyl, C₁₋₄alkoxy, —C₃₋₆cycloalkyl, —OC₃₋₆cycloalkyl,—OCH₂Ph, —CF₃, —OCF₃, —SCF₃, —(C═O)R^(k) (wherein R^(k) is H, C₁₋₄alkyl,—OH, phenyl, benzyl, phenethyl or C₁₋₆alkoxy), —(N—R^(t))(C═O)R^(k)(where R^(t) is H or C₁₋₄alkyl), —(N—R^(t))SO₂C₁₋₄alkyl,—(S═(O)_(p))—C₁₋₄alkyl (wherein p is 0, 1 or 2), nitro, —SO₂NR^(l)R^(m)(wherein R^(l) and R^(m) are independently selected from H, C₁₋₄alkyl,phenyl, benzyl or phenethyl, or R^(l) and R^(m) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₄alkyl), —(C═O)NR^(l)R^(m), cyano or phenyl, where any phenyl oralkyl or cycloalkyl moiety of the foregoing is optionally andindependently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; and enantiomers,diastereomers and pharmaceutically acceptable salts and esters thereof,with the following provisos, that R⁶ adjacent to N must be H where R⁴adjacent to N is other than H, that R⁷ is not —CH₂CH₂OH; and that wherethe core molecule is a 4H-furo, then one of R⁴ and R⁶ adjacent to N mustnot be methyl when the other is hydrogen unless R⁶ and R⁴ are takentogether to form a bridging moiety.
 2. A pharmaceutical compositioncontaining at least one compound of formula (I)

Y is O; Z is O or S; n is 1 or 2; m is 1 or 2; n+m is 2 or 3; R¹ is H orC₁₋₆alkyl; R² is H, F, Cl, Br or C₁₋₆alkyl; R³ and R⁴ are,independently, H, C₁₋₄alkyl, C₃₋₆cycloalkyl, C₁₋₄alkyl(C₃₋₆cycloalkyl),cyano, —CF₃, —(CO)NR^(p)R^(q), —(CO)OR^(r), —CH₂NR^(p)R^(q) Or—CH₂OR^(r); where R^(p), R^(q) and R^(r) are independently selected fromH, C₁₋₄alkyl, C₃₋₆cycloalkyl, phenyl, —C₁₋₂alkyl(C₃₋₆cycloalkyl), benzylor phenethyl, or R^(p) and R^(q) taken together with the nitrogen towhich they are attached, form a 4-7 membered heterocyclic ring with 0 or1 additional heteroatoms selected from O, S, NH or NC₁₋₆alkyl, and whereany phenyl or alkyl or cycloalkyl moiety of the foregoing is optionallyand independently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; R⁵ and R⁶ are,independently, H or C₁₋₆alkyl; R⁷ is —R^(a), —R^(b)R^(a), —R^(e)—O—R^(a)or —R^(e)—N(R^(c))(R^(d)), where R^(a) is H, cyano,—(C═O)N(R^(c))(R^(d)), —C(═NH)(NH₂), C₁₋₁₀alkyl, C₂₋₈alkenyl,C₃₋₈cycloalkyl, C₄₋₇heterocyclic radical or phenyl, where theC₄₋₇heterocyclic radical is attached at a carbon atom and contains oneof O, S, NH or NC₁₋₄alkyl, and optionally an additional NH or NC₁₋₆alkylin rings of 5 or 6 or 7 members, where R^(b) is C₁₋₈alkylene orC₂₋₈alkenylene, where R^(e) is C₂₋₈alkylene or C₂₋₈alkenylene, whereR^(c) and R^(d) are each independently H, C₁₋₄alkyl, C₂₋₄alkenyl,C₃₋₆cycloalkyl or phenyl, or R^(c) and R^(d) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₆alkyl, and where any phenyl or alkyl or cycloalkyl moiety of theforegoing is optionally and independently substituted with between 1 and3 substituents selected from C₁₋₃alkyl, halo, hydroxy, amino, andC₁₋₃alkoxy; alternatively, R⁷ may be taken together with an adjacent R⁴as well as their carbon and nitrogen of attachment to form a 5, 6 or 7membered heterocyclic ring, with 0 or 1 additional heteroatoms selectedfrom O, S, NH or NC₁₋₆alkyl, and optionally and independentlysubstituted with between 1 and 3 substituents selected from C₁₋₃alkyl,halo, hydroxy, amino, and C₁₋₃alkoxy; R⁸ and R⁹ are, independently, H,F, Cl, Br, I, C₁₋₄alkyl, C₁₋₄alkoxy, —C₃₋₆cycloalkyl, —OC₃₋₆cycloalkyl,—OCH₂Ph, —CF₃, —OCF₃, —SCF₃, —(C═O)R^(k) (wherein R^(k) is H, C₁₋₄alkyl,—OH, phenyl, benzyl, phenethyl or C₁₋₆alkoxy), —(N—R^(t))(C═O)R^(k)(where R^(t) is H or C₁₋₄alkyl), —(N—R^(t))SO₂C₁₋₄alkyl,—(S═(O)_(p))—C₁₋₄alkyl (wherein p is 0, 1 or 2), nitro, —SO₂NR^(l)R^(m)(wherein R^(l) and R^(m) are independently selected from H, C₁₋₄alkyl,phenyl, benzyl or phenethyl, or R^(l) and R^(m) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₄alkyl), —(C═O)NR^(l)R^(m), cyano or phenyl, where any phenyl oralkyl or cycloalkyl moiety of the foregoing is optionally andindependently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; and enantiomers,diastereomers and pharmaceutically acceptable salts and esters thereof,with the following provisos, that R⁶ adjacent to N must be H where R⁴adjacent to N is other than H, that R⁷ is not —CH₂CH₂OH; and that wherethe core molecule is a 4H-furo, then one of R⁴ and R⁶ adjacent to N mustnot be methyl when the other is hydrogen unless R⁶ and R⁴ are takentogether to form a bridging moiety.
 3. A method for modulating the H₄receptor, comprising exposing said H₄ receptor to an effective amount ofat least one compound of claim
 1. 4. A compound selected from the groupconsisting of(4H-Furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;(2-Methyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;(2,3-Dimethyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;(2-Methyl-4H-furo[3,2-b]pyrrol-5-yl)-piperazin-1-yl-methanone; andenantiomers, diastereomers and pharmaceutically acceptable salts andesters thereof.
 5. A method for modulating the H₄ receptor, comprisingexposing said H₄ receptor to an effective amount of at least onecompound of claim
 4. 6. A pharmaceutical composition containing at leastone compound of claim 4.